Pheromones and the Luteinizing Hormone for Inducing Proliferation of Neural Stem Cells and Neurogenesis

ABSTRACT

The present invention provides a method of increasing neural stem cell numbers or neurogenesis by using a pheromone, a luteinizing hormone (LH) and/or a human chorionic gonadotrophin (hCG). The method can be practiced in vivo to obtain more neural stem cells in situ, which can in turn produce more neurons or glial cells to compensate for lost or dysfunctional neural cells. The method can also be practiced in vitro to produce a large number of neural stem cells in culture. The cultured stem cells can be used, for example, for transplantation treatment of patients or animals suffering from or suspected of having neurodegenerative diseases or conditions.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/544,915, filed Feb. 13, 2004, under 35 U.S.C. §119(e). The entiredisclosure of the prior application is hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to methods of increasing neural stem cell numbersand neurogenesis, as well as compositions useful therefore.

REFERENCES

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All of the publications, patents and patent applications cited above orelsewhere in this application are herein incorporated by reference intheir entirety to the same extent as if the disclosure of eachindividual publication, patent application or patent was specificallyand individually indicated to be incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

In recent years, neurodegenerative disease has become an importantconcern due to the expanding elderly population which is at greatestrisk for these disorders. Neurodegenerative diseases include thediseases which have been linked to the degeneration of neural cells inparticular locations of the central nervous system (CNS), leading to theinability of these cells to carry out their intended function. Thesediseases include Alzheimer's Disease, Huntington's Disease, AmyotrophicLateral Sclerosis, and Parkinson's Disease. In addition, probably thelargest area of CNS dysfunction (with respect to the number of affectedpeople) is not characterized by a loss of neural cells but rather byabnormal functioning of existing neural cells. This may be due toinappropriate firing of neurons, or the abnormal synthesis, release, andprocessing of neurotransmitters. These dysfunctions may be the result ofwell studied and characterized disorders such as depression andepilepsy, or less understood disorders such as neurosis and psychosis.Moreover, brain injuries often result in the loss of neural cells, theinappropriate functioning of the affected brain region, and subsequentbehavior abnormalities.

Consequently, it is desirable to supply neural cells to the brain tocompensate for degenerate or lost neurons in order to treatneurodegenerative diseases or conditions. One approach to this end is totransplant neural cells into the brain of the patient. This approachrequires a source of large amounts of neural cells, preferably from thesame individual or a closely related individual such thathost-versus-graft or graft-versus-host rejections can be minimized. Asit is not practical to remove a large amount of neurons or glial cellsfrom one person to transplant to another, a method to culture largequantity of neural cells is necessary for the success of this approach.

Another approach is to induce the production of neural cells in situ tocompensate for the lost or degenerate cells. This approach requiresextensive knowledge about whether it is possible to produce neural cellsin brains, particularly adult brains, and how.

The development of techniques for the isolation and in vitro culture ofmultipotent neural stem cells (for example, see U.S. Pat. Nos.5,750,376; 5,980,885; 5,851,832) significantly increased the outlook forboth approaches. It was discovered that fetal brains can be used toisolate and culture multipotent neural stem cells in vitro. Moreover, incontrast to the long time belief that adult brain cells are not capableof replicating or regenerating brain cells, it was found that neuralstem cells may also be isolated from brains of adult mammals. These stemcells, either from fetal or adult brains, are capable ofself-replicating. The progeny cells can again proliferate ordifferentiate into any cell in the neural cell lineage, includingneurons, astrocytes and oligodendrocytes. Therefore, these findings notonly provide a source of neural cells which can be used intransplantations, but also demonstrate the presence of multipotentneural stem cells in adult brain and the possibility of producingneurons or glial cells from these stem cells in situ.

It is therefore desirable to develop methods of efficiently producingneural stem cells for two purposes: to obtain more stem cells and henceneural cells which can be used in transplantation therapies, and toidentify methods which can be used to produce more stem cells in situ.

SUMMARY OF THE INVENTION

The present invention provides a method of increasing neural stem cellnumbers by using a pheromone, a luteinizing hormone (LH) or humanchorionic gonadotrophin (hCG). The method can be practiced in vivo toobtain more neural stem cells in situ, which can in turn produce moreneurons or glial cells to compensate for lost or dysfunctional neuralcells. The method can also be practiced in vitro to produce a largenumber of neural stem cells in culture. The cultured stem cells can beused, for example, for transplantation treatment of patients or animalssuffering from or suspected of having neurodegenerative diseases orconditions.

Accordingly, one aspect of the present invention provides a method ofincreasing neural stem cell number, comprising providing an effectiveamount of a pheromone, an LH or hCG to at least one neural stem cellunder conditions which result in an increase in the number of neuralstem cells. The neural stem cell may be located in the brain of amammal, in particular in the subventricular zone of the brain of themammal. Alternatively, the neural stem cell may be located in thehippocampus of the mammal. Although mammals of all ages can be subjectedto this method, it is preferable that the mammal is not an embryo. Morepreferably, the mammal is an adult.

The mammal may suffer from or be suspected of having a neurodegenerativedisease or condition. The disease or condition may be a spinal cordinjury or brain injury, such as stroke or an injury caused by a surgery.The disease or condition may be aging, which is associated with asignificant reduction in the number of neural stem cells. The disease orcondition can also be a neurodegenerative disease, particularlyAlzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis, or Parkinson's disease.

Alternatively, the neural stem cell may be in a culture in vitro. Whenpracticed in vitro, it is preferable that LH or hCG is used instead ofpheromones.

The pheromone can be any pheromone that is capable of increasing neuralstem cell numbers in the mammal. Assays for determining if a substanceis capable of increasing neural stem cell numbers are established in theart and described herein (e.g., see Examples 1 and 3). The pheromone ispreferably selected from the group consisting of2-sec-butyl-4,5-dihydrothiazole (SBT), 2,3-dehydro-exo-brevicomin (DHB),alpha and beta farnesenes, 6-hydroxy-6-methyl-3-heptanone, 2-heptanone,trans-5-hepten-2-one, trans-4-hepten-2-one, n-pentyl acetate,cis-2-penten-1-yl-acetate, 2,5-dimethylpyrazine, dodecyl propionate, and(Z)-7-dodecen-1-yl acetate.

Whether the pheromone, LH or hCG is used in vivo or in vitro, otheragents may be applied in combination, such as follicle-stimulatinghormone (FSH), gonadotropin releasing hormone (GnRH), prolactin,prolactin releasing peptide (PRP) erythropoietin, cyclic AMP, pituitaryadenylate cyclase activating polypeptide (PACAP), serotonin, bonemorphogenic protein (BMP), epidermal growth factor (EGF), transforminggrowth factor alpha (TGFalpha), transforming growth factor beta(TGFbeta), fibroblast growth factor (FGF), estrogen, growth hormone,growth hormone releasing hormone, insulin-like growth factors, leukemiainhibitory factor, ciliary neurotrophic factor (CNTF), brain derivedneurotrophic factor (BDNF), thyroid hormone, thyroid stimulatinghormone, sonic hedgehog (SHH), and/or platelet derived growth factor(PDGF). The LH or hCG may be any LH or hCG analog or variant which hasthe activity of the native LH or hCG.

Another aspect of the present invention provides a method of treating orameliorating a neurodegenerative disease or condition in a mammal,comprising providing an effective amount of a pheromone, LH or hCG tothe brain of the mammal. The disease or condition may be a CNS injury,such as stroke or an injury caused by a brain/spinal cord surgery. Thedisease or condition may be aging, which is associated with asignificant reduction in the number of neural stem cells. The disease orcondition can also be a neurodegenerative disease, particularlyAlzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis, or Parkinson's disease.

The mammal can optionally receive a transplantation of neural stem cellsand/or neural stem cell progeny. The transplantation may take placebefore, after, or at the same time the mammal receives the pheromone, LHor hCG. Preferably, the mammal receives the transplantation prior to orconcurrently with the pheromone, LH or hCG.

The mammal can optionally receive at least one additional agent, such aserythropoietin, cyclic AMP, pituitary adenylate cyclase activatingpolypeptide (PACAP), serotonin, bone morphogenic protein (BMP),epidermal growth factor (EGF), transforming growth factor alpha(TGF.alpha.), fibroblast growth factor (FGF), estrogen, growth hormone,insulin-like growth factor 1, and/or ciliary neurotrophic factor (CNTF).

The pheromone, LH/hCG and/or the additional agent can be provided by anymethod established in the art. For example, they can be administeredintravascularly, intrathecally, intravenously, intramuscularly,subcutaneously, intraperitoneally, topically, orally, rectally,vaginally, nasally, by inhalation or into the brain. The administrationis preferably performed systemically, particularly by subcutaneousadministration. The pheromone, LH/hCG or additional agent can also beprovided by administering to the mammal an effective amount of an agentthat can increase the amount of endogenous pheromone, LH/hCG or theadditional agent in the mammal. For example, the level of LH in ananimal can be increased by using GnRH.

When the pheromone, LH/hCG or the additional agent is not directlydelivered into the brain, a blood brain barrier permeabilizer can beoptionally included to facilitate entry into the brain. Blood brainbarrier permeabilizers are known in the art and include, by way ofexample, bradykinin and the bradykinin agonists described in U.S. Pat.Nos. 5,686,416; 5,506,206 and 5,268,164 (such asNH₂-arginine-proline-hydroxyproxyproline-glycine-thienylalanine-serine-proline-4-Me-tyrosine.psi.(—CH₂NH)-arginine-COOH).Alternatively, the molecules to be delivered can be conjugated to thetransferrin receptor antibodies as described in U.S. Pat. No. 6,329,508;6,015,555; 5,833,988 or 5,527,527. The molecules can also be deliveredas a fusion protein comprising the molecule and a ligand that isreactive with a brain capillary endothelial cell receptor, such as thetransferrin receptor (see, e.g., U.S. Pat. No. 5,977,307).

Another aspect of the present invention provides a method of enhancingneuron formation from neural stem cells, comprising providing apheromone, LH or hCG to at least one neural stem cell under conditionsthat result in enhanced neuron formation from said neural stem cell.Further provided is a method of increasing new neuron formation in theolfactory bulb of a mammal, comprising providing an effective amount ofa pheromone, LH or hCG to the mammal. Compositions and pharmaceuticalcompositions comprising a pheromone, LH or hCG, and at least oneadditional agent are also provided.

Also provided are cellular compositions prepared according to thepresent invention. In particular, neural stem cell cultures that havebeen exposed to LH/hCG are provided. These cultures have higher levelsof neural stem cells and/or neurons, and can be used, for example, fortransplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The effects of male odors on proliferation of neural stem cellsin the SVZ of female mice after an exposure of 2, 7 or 14 days. 2 D, 7 Dand 14 D indicate an exposure of 2, 7 and 14 days, respectively. F-M,female mice exposed to male odors; F-F, female mice exposed to femaleodors. The raw data are shown on the top of each panel.

-   -   (A) shows the effects on the number of BrdU positive cells in        the SVZ.    -   (B) shows the effects on the number of Ki67 positive cells in        the SVZ.    -   (C) shows the comparison of littermates and non-littermates.

FIG. 2. The effects of female odors on proliferation of neural stemcells in the SVZ of male mice after an exposure of 2, 7 or 14 days. 2 D,7 D and 14 D indicate an exposure of 2, 7 and 14 days, respectively.M-F, male mice exposed to female odors; M-M, male mice exposed to maleodors. The raw data are shown on the top of each panel.

-   -   (A) shows the effects on the number of BrdU positive cells in        the SVZ.    -   (B) shows the effects on the number of Ki67 positive cells in        the SVZ.    -   (C) shows the comparison of littermates and non-littermates.

FIG. 3. The effects of male odors on proliferation of neural stem cellsin the hippocampus of female mice after an exposure of 2, 7 or 14 days.2 D, 7 D and 14 D indicate an exposure of 2, 7 and 14 days,respectively. F-M, female mice exposed to male odors; F-F, female miceexposed to female odors. The raw data are shown on the top of eachpanel.

FIG. 4. The effects of male odors on neurogenesis in female mice afteran exposure of 2, 7 or 14 days. 2 D, 7 D and 14 D indicate an exposureof 2, 7 and 14 days, respectively. F-M, female mice exposed to maleodors; F-F, female mice exposed to female odors. DCX, doublecortin. Theraw data are shown on the top of each panel.

FIG. 5. The effects of female odors on neurogenesis in male mice afteran exposure of 2, 7 or 14 days. 2 D, 7 D and 14 D indicate an exposureof 2, 7 and 14 days, respectively. M-F, male mice exposed to femaleodors; M-M, male mice exposed to male odors. DCX, doublecortin. The rawdata are shown on the top of each panel.

FIG. 6. The TUNEL (terminal deoxynucleotidyltransferase-mediated dUTPnick end labeling) assay. Female mice were exposed to male odors (F-M)or female odors (F-F) for 7 days, and the number of cells that underwentprogrammed cell death was determined by the TUNEL assay. (A) and (B)show the apoptotic cell counts in the SVZ and olfactory bulb,respectively.

FIG. 7. The effects of LH on the number of BrdU positive cells in theSVZ in female mice. (A) and (B) show the effects of LH after a 2-dayinfusion (A) or 6-day infusion (B) of LH, respectively. VEH, vehicle.

FIG. 8. The effects of LH on the number of BrdU positive cells in theSVZ in male mice after a 2-day infusion of LH. VEH, vehicle.

FIG. 9. The effects of LH receptors in pheromone-induced neural stemcell proliferation in female mice. (A) and (B) show the effects of LHreceptor knock-out in the SVZ (A) and hippocampus (B), respectively.(−/−): LH receptor knock-out. (+/+): wild-type. Baseline: mice exposedto unodorized cages. Female-Female: female mice exposed to female odors.Female-Male: female mice exposed to male odors. P*<0.05; LSD posthoctest.

FIG. 10. The effects of LH receptors in pheromone-induced neural stemcell proliferation in male mice. (A) and (B) show the effects of LHreceptor knock-out in the SVZ (A) and hippocampus (B), respectively.(−/−): LH receptor knock-out. (+/+): wild-type. Baseline: mice exposedto unodorized cages. Male-Female: male mice exposed to female odors.Male-Female: male mice exposed to female odors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of increasing neural stem cellnumbers or neurogenesis by using a pheromone, a luteinizing hormone (LH)or a human chorionic gonadotrophin (hCG). The method can be practiced invivo to obtain more neural stem cells in situ, which can in turn producemore neurons or glial cells to compensate for lost or dysfunctionalneural cells. The method can also be practiced in vitro to produce alarge number of neural stem cells in culture. The cultured stem cellscan be used, for example, for transplantation treatment of patients oranimals suffering from or suspected of having neurodegenerative diseasesor conditions.

Prior to describing the invention in further detail, the terms used inthis application are defined as follows unless otherwise indicated.

DEFINITIONS

A “neural stem cell” is a stem cell in the neural cell lineage. A stemcell is a cell which is capable of reproducing itself. In other words,daughter cells which result from stem cell divisions include stem cells.The neural stem cells are capable of ultimately differentiating into allthe cell types in the neural cell lineage, including neurons, astrocytesand oligodendrocytes (astrocytes and oligodendrocytes are collectivelycalled glia or glial cells). Thus, the neural stem cells referred toherein are multipotent neural stem cells.

A “neurosphere” or “sphere” is a group of cells derived from a singleneural stem cell as the result of clonal expansion. A “primaryneurosphere” refers to a neurosphere generated by plating as primarycultures brain tissue which contains neural stem cells. The method forculturing neural stem cells to form neurospheres has been described in,for example, U.S. Pat. No. 5,750,376. A “secondary neurosphere” refersto a neurosphere generated by dissociating primary neurospheres andallowing the individual dissociated cells to form neurospheres again.

A polypeptide which shares “substantial sequence similarity” with anative factor is at least about 30% identical with the native factor atthe amino acid level. The polypeptide is preferably at least about 40%,more preferably at least about 60%, yet more preferably at least about70%, and most preferably at least about 80% identical with the nativefactor at the amino acid level.

The phrase “percent identity” or “% identity” of an analog or variantwith a native factor refers to the percentage of amino acid sequence inthe native factor which are also found in the analog or variant when thetwo sequences are aligned. Percent identity can be determined by anymethods or algorithms established in the art, such as LALIGN or BLAST.

A polypeptide possesses a “biological activity” of a native factor if itis capable of binding to the receptor for the native factor or beingrecognized by a polyclonal antibody raised against the native factor.Preferably, the polypeptide is capable of specifically binding to thereceptor for the native factor in a receptor binding assay.

A “functional agonist” of a native factor is a compound that binds toand activates the receptor of the native factor, although it does notnecessarily share a substantial sequence similarity with the nativefactor.

An “LH” is a protein which (1) comprises a polypeptide that sharessubstantial sequence similarity with a native mammalian LH, preferablythe native human LH; and (2) possesses a biological activity of thenative mammalian LH. The native mammalian LH is a gonadotropin secretedby the anterior lobe of the pituitary. LH is a heterodimer consisting ofnon-covalently bound alpha and beta subunits. The alpha subunit iscommon among LH, FSH and hCG, and the beta subunit is specific for eachhormone. The LH useful in the present invention may have the nativealpha subunit, with the beta subunit sharing a substantial sequencesimilarity with a native mammalian LH. Alternatively, the LH may havethe native beta subunit, with the alpha subunit sharing a substantialsequence similarity with a native mammalian LH. The LH may also haveboth the alpha and beta subunit sharing a substantial sequencesimilarity with a native, corresponding subunit. Thus, the term “LH”encompasses LH analogs which comprise a deletional, insertional, orsubstitutional mutants of a native LH subunit. Furthermore, the term“LH” encompasses the LHs from other species and the naturally occurringvariants thereof. In addition, an “LH” may also be a functional agonistof a native mammalian LH receptor.

An “hCG” is a protein which (1) comprises a polypeptide that sharessubstantial sequence similarity with the native hCG; and (2) possesses abiological activity of the native hCG. The native hCG is a heterodimerconsisting of non-covalently bound alpha and beta subunits. The alphasubunit is common among LH, FSH and hCG, and the beta subunit isspecific for each hormone. However, the beta subunits of hCG and LHshares a 85% sequence similarity. The hCG useful in the presentinvention may have the native alpha subunit, with the beta subunitsharing a substantial sequence similarity with the native hCG.Alternatively, the hCG may have the native beta subunit, with the alphasubunit sharing a substantial sequence similarity with the native hCG.The hCG may also have both the alpha and beta subunit sharing asubstantial sequence similarity with the native, corresponding subunit.Thus, the term “hCG” encompasses hCG analogs which comprise adeletional, insertional, or substitutional mutants of a native hCGsubunit. Furthermore, the term “hCG” encompasses the hCG counterpartsfrom other species and the naturally occurring variants thereof. Inaddition, an “hCG” may also be a functional agonist of a nativemammalian hCG/LH receptor.

A “prolactin” is a polypeptide which (1) shares substantial sequencesimilarity with a native mammalian prolactin, preferably the nativehuman prolactin; and (2) possesses a biological activity of the nativemammalian prolactin. The native human prolactin is a 199-amino acidpolypeptide synthesized mainly in the pituitary gland. Thus, the term“prolactin” encompasses prolactin analogs which are the deletional,insertional, or substitutional mutants of the native prolactin.Furthermore, the term “prolactin” encompasses the prolactins from otherspecies and the naturally occurring variants thereof.

In addition, a “prolactin” may also be a functional agonist of a nativemammalian prolactin receptor. For example, the functional agonist may bean activating amino acid sequence disclosed in U.S. Pat. No. 6,333,031for the prolactin receptor; a metal complexed receptor ligand withagonist activities for the prolactin receptor (U.S. Pat. No. 6,413,952);G120RhGH, which is an analog of human growth hormone but acts as aprolactin agonist (Mode et al., 1996); or a ligand for the prolactinreceptor as described in U.S. Pat. Nos. 5,506,107 and 5,837,460.

An “EGF” means a native EGF or any EGF analog or variant that shares asubstantial amino acid sequence similarity with a native EGF, as well asat least one biological activity with the native EGF, such as binding tothe EGF receptor. Particularly included as an EGF is the native EGF ofany species, TGF.alpha., or recombinant modified EGF. Specific examplesinclude, but are not limited to, the recombinant modified EGF having adeletion of the two C-terminal amino acids and a neutral amino acidsubstitution at position 51 (particularly EGF51 gln51; U.S. PatentApplication Publication No. 20020098178A1), the EGF mutein (EGF-X.sub.6)in which the His residue at position 16 is replaced with a neutral oracidic amino acid (U.S. Pat. No. 6,191,106), the 52-amino acid deletionmutant of EGF which lacks the amino terminal residue of the native EGF(EGF-D), the EGF deletion mutant in which the N-terminal residue as wellas the two C-terminal residues (Arg-Leu) are deleted (EGF-B), the EGF-Din which the Met residue at position 21 is oxidized (EGF-C), the EGF-Bin which the Met residue at position 21 is oxidized (EGF-A),heparin-binding EGF-like growth factor (HB-EGF), betacellulin,amphiregulin, neuregulin, or a fusion protein comprising any of theabove. Other useful EGF analogs or variants are described in U.S. PatentApplication Publication No. 20020098178A1, and U.S. Pat. Nos. 6,191,106and 5,547,935.

In addition, an “EGF” may also be a functional agonist of a nativemammalian EGF receptor. For example, the functional agonist may be anactivating amino acid sequence disclosed in U.S. Pat. No. 6,333,031 forthe EGF receptor, or an antibody that has agonist activities for the EGFreceptor (Fernandez-Pol, 1985 and U.S. Pat. No. 5,723,115).

A “PACAP” means a native PACAP or any PACAP analog or variant thatshares a substantial amino acid sequence similarity with a native PACAP,as well as at least one biological activity with the native PACAP, suchas binding to the PACAP receptor. Useful PACAP analogs and variantsinclude, without being limited to, the 38 amino acid and the 27 aminoacid variants of PACAP (PACAP38 and PACAP27, respectively), and theanalogs and variants disclosed in, e.g., U.S. Pat. Nos. 5,128,242;5,198,542; 5,208,320; 5,326,860; 5,623,050; 5,801,147 and 6,242,563.

In addition, a “PACAP” may also be a functional agonist of a nativemammalian PACAP receptor. For example, the functional agonist may bemaxadilan, a polypeptide that acts as a specific agonist of the PACAPtype-1 receptor (Moro et al., 1997).

An “erythropoietin (EPO)” means a native EPO or any EPO analog orvariant that shares a substantial amino acid sequence similarity with anative EPO, as well as at least one biological activity with the nativeEPO, such as binding to the EPO receptor. Erythropoietin analogs andvariants are disclosed, for example, in U.S. Pat. Nos. 6,048,971 and5,614,184.

In addition, an “EPO” may also be a functional agonist of a nativemammalian EPO receptor. For example, the functional agonist may be EMP1(EPO mimetic peptide 1, Johnson et al., 2000); one of the short peptidemimetics of EPO as described in Wrighton et al., 1996 and U.S. Pat. No.5,773,569; any small molecular EPO mimetic as disclosed in Kaushansky,2001; an antibody that activates the EPO receptor as described in U.S.Pat. No. 5,885,574, WO 96/40231, WO 97/48729, Fernandez-Pol, 1985 orU.S. Pat. No. 5,723,115; an activating amino acid sequence as disclosedin U.S. Pat. No. 6,333,031 for the EPO receptor; a metal complexedreceptor ligand with agonist activities for the EPO receptor (U.S. Pat.No. 6,413,952), or a ligand for the EPO receptor as described in U.S.Pat. Nos. 5,506,107 and 5,837,460.

A “LH/hCG-inducing agent” is a substance that, when given to an animal,is capable of increasing the amount of LH or hCG in the animal. Forexample, LH releasing hormone (LHRH) stimulates the secretion of LH.

A “pheromone” is a substance that serves as a signal to another animalof the same species, usually of the opposite gender. A mammalianpheromone can be a protein a small molecule. Preferably, the pheromoneis selected from the group consisting of 2-sec-butyl-4,5-dihydrothiazole(SBT), 2,3-dehydro-exo-brevicomin (DHB), alpha and beta farnesenes,6-hydroxy-6-methyl-3-heptanone, 2-heptanone, trans-5-hepten-2-one,trans-4-hepten-2-one, n-pentyl acetate, cis-2-penten-1-yl-acetate,2,5-dimethylpyrazine, dodecyl propionate, and (Z)-7-dodecen-1-yl acetate(see, e.g., Dulac et al., 2003).

“Enhancing” the formation of a cell type means increasing the number ofthe cell type. Thus, an agent can be used to enhance neuron formation ifthe number of neurons in the presence of the agent is larger than thenumber of neurons absent the agent. The number of neurons in the absenceof the agent may be zero or more.

A “neurodegenerative disease or condition” is a disease or medicalcondition associated with neuron loss or dysfunction. Examples ofneurodegenerative diseases or conditions include neurodegenerativediseases, CNS injuries or CNS dysfunctions. Neurodegenerative diseasesinclude, for example, Alzheimer's disease, macular degeneration,glaucoma, diabetic retinopathy, peripheral neuropathy, Huntington'sdisease, amyotrophic lateral sclerosis, and Parkinson's disease. CNSinjuries include, for example, stroke (e.g., hemorrhagic stroke, focalischemic stroke or global ischemic stroke) and traumatic brain or spinalcord injuries (e.g. injuries caused by a brain or spinal cord surgery orphysical accidents). CNS dysfunctions include, for example, depression,epilepsy, neurosis and psychosis.

“Treating or ameliorating” means the reduction or complete removal ofthe symptoms of a disease or medical condition.

A mammal “suspected of having a neurodegenerative disease or condition”is a mammal which is not officially diagnosed with the neurodegenerativedisease or condition but shows a symptom of the neurodegenerativedisease or condition, is susceptible to the neurodegenerative disease orcondition due to family history or genetic predisposition, or haspreviously had the neurodegenerative disease or condition and is subjectto the risk of recurrence.

“Transplanting” a composition into a mammal refers to introducing thecomposition into the body of the mammal by any method established in theart. The composition being introduced is the “transplant”, and themammal is the “recipient”. The transplant and the recipient may besyngeneic, allogeneic or xenogeneic. Preferably, the transplantation isan autologous transplantation.

An “effective amount” is an amount of a therapeutic agent sufficient toachieve the intended purpose. For example, an effective amount of an LHor hCG to increase the number of neural stem cells is an amountsufficient, in vivo or in vitro, as the case may be, to result in anincrease in neural stem cell number. An effective amount of an LH or hCGto treat or ameliorate a neurodegenerative disease or condition is anamount of the LH/hCG sufficient to reduce or remove the symptoms of theneurodegenerative disease or condition. The effective amount of a giventherapeutic agent will vary with factors such as the nature of theagent, the route of administration, the size and species of the animalto receive the therapeutic agent, and the purpose of the administration.The effective amount in each individual case may be determinedempirically by a skilled artisan according to established methods in theart.

Methods

Neural stem cells are located in two regions of the adult mammalianbrain (Reynolds and Weiss, 1992): the dendate gyrus of the hippocampusand the subventricular zone (SVZ) of the lateral ventricles (Luskin1993; Menezes et al., 1995; Frisen et al., 1998; Peretto et al., 1999;Gage, 2000; Rochefort et al., 2002). Neural stem cells follow twomitotic pathways that contribute to their growth and proliferation. Thefirst mitotic path is where neural stem cells divide symmetrically as ameans of regeneration and self-renewal. The second mitotic division isasymmetrical, which results in a daughter neural stem cell and aprogenitor cell. It is ultimately the progenitor cell that takes on aterminalistic fate as one of the cell types of the central nervoussystem. For example, in the case of neurogenesis, it is the neuronalprogenitor cell that gives rise to a neuron (Weiss et al., 1996;Morrison and Shah, 1997; Peretto et al., 1999; Rao, 1999).

The neuronal progenitors of the hippocampus reside in the dentate gyrusand have the ability to proliferate and migrate to the granular celllayer to differentiate into granule cells (Nilsson et al., 1999; Gage2000; Rochefort et al., 2002). In the SVZ, neural stem cells andprogenitors proliferate, then the progenitors follow a migratory path,known as the rostral migratory stream (RMS), where they are destined forthe olfactory bulb (OB) to become interneurons (Luskin 1993; Menezes etal., 1995; Rao, 1999; Rochefort et al., 2002).

It has been shown that an enriched olfactory environment, created withnovel odors, increased neurogenesis in the olfactory bulb and improvedodor memory (Rochefort et al., 2002). Although the olfactory bulbinterneurons are derived from the neural stem cells in the SVZ, exposureto the enriched olfactory environment had no effect on cellproliferation in the SVZ (Rochefort et al., 2002). As described in thepresent invention, however, we observed the surprising effects of maleand female odors on the opposite gender in neural stem cellproliferation and neurogenesis.

To determine the impact of male or female odors, adult mice were exposedto the odors of the opposite gender for 2 days, 7 days or 14 days. Acontrol group was exposed to the odors of the same gender for the sameperiod of time. The mice then received BrdU to label proliferatingcells, and the locations of the BrdU positive cells were identified byimmunohistochemical studies (Example 1). As shown in FIG. 1A,proliferating cells in the SVZ of female mice remained at the same levelafter being exposed to female odors for 2, 7 or 14 days. In the femalegroup exposed to male odors, however, proliferating cells in the SVZchanged with time: increased significantly after 7 days and decreasedsignificantly after 14 days. A 2-day exposure had no significant effect.The same pattern was observed when Ki67 was used to label proliferatingcells (FIG. 1B), indicating that the change in BrdU positive cellsreflected a change of proliferation level.

Female odors also affected proliferation in male mouse brains, but in adifferent temporal pattern. When males were exposed to female odors for2 days, there was a sudden increase in the number of BrdU positive cells(FIG. 2A) or Ki67 positive cells (FIG. 2B). After a 7 or 14 dayexposure, however, the number of newly proliferated cells decreased tothe control level.

Strikingly, the neural stem cells in the hippocampus also responded togender-specific odors. Again, exposure for two days to male odors had nosignificant effects on female mice, but a 7-day exposure resulted in asignificant increase in proliferation in the hippocampus (FIG. 3). Afteran exposure for 14 days, levels of proliferating cells weresignificantly lower in females exposed to male odors when compared withthe females that had been exposed to female odors. To our knowledge,this is the first time that any stimulus, other than growth factors(e.g., EGF plus FGF), has been shown to exert the same effects on theneural stem cells in the SVZ and the hippocampus. Usually the effectsare opposite. For example, prolactin affects the SVZ but not thehippocampus (Shingo et al., 2003); estrogen stimulates proliferation inthe hippocampus but not in the SVZ (Tanapat et al., 1999); an enrichedenvironment and physical activities promote hippocampal neurogenesis,but not SVZ neurogenesis (Brown et al., 2003).

Neurogenesis was also enhanced upon exposure to the odors of theopposite gender (Example 2). Thus, tissue sections from the animalsdescribed above were stained for doublecortin, a cytoplasmic proteinexpressed in neuronal progenitor cells, to determine the extent ofneurogenesis in the mice described above. As in the case ofproliferating cells, female mice had significantly more doublecortinpositive cells after a 7-day exposure to male odors (FIG. 4) while malemice had significantly more doublecortin positive cells after a 2-dayexposure to female odors (FIG. 5).

To determine if pheromones from the opposite gender also impact survivalof neural cells, the TUNEL assay was performed. The results indicatethat no significant difference can be observed in the SVZ (FIG. 6A) orolfactory bulb (FIG. 6B) of female mice after a 7-day exposure to maleodors.

Male pheromones are known to increase the levels of the luteinizinghormone (LH) and decrease the levels of prolactin, while femalepheromones are associated with an increase in prolactin (Dulac et al.,2003). In an attempt to investigate how pheromones enhance neural stemcell proliferation and neurogenesis in the opposite gender, animals wereinfused with LH. The results show that LH increase proliferationsignificantly in the SVZ of both female (FIGS. 7A and 7B) and male mice(FIG. 8). Consistent with these results, LH is also capable ofincreasing self-renewal of neural stem cells in culture (Example 3).

Accordingly, the present invention provides a method of increasingneural stem cells numbers either in vivo or in vitro using a pheromoneand/or LH. Human chorionic gonadotrophin (hCG) is expected to have thesame effect as LH as hCG is an analog of, and shares the same receptorwith, LH. When used to increase neural stem cell number in vivo, thismethod will result in a larger pool of neural stem cells in the brain.This larger pool of neural stem cells can subsequently generate moreneural cells, particularly neurons or glial cells, than would apopulation of stem cells without pheromone, LH/hCG. The neural cells, inturn, can compensate for lost or degenerate neural cells which areassociated with neurodegenerative diseases and conditions, includingnervous system injuries.

LH/hCG or other factors induced by pheromones can also be used toincrease neural stem cell numbers in vitro. The resulting stem cells canbe used to produce more neurons and/or glial cells in vitro, or used intransplantation procedures into humans or animals suffering fromneurodegenerative diseases or conditions. It is preferable that neuralstem cells produced according to the present invention, rather thanneurons or glial cells, are transplanted. Once neural stem cells aretransplanted, growth and/or differentiation agents can be administeredin vivo to further increase the number of stem cells, or to selectivelyenhance neuron formation or glial cell formation. The additional agentscan likewise be used in vitro with LH or hCG, or administered in vivo incombination with pheromone/LH/hCG.

Exemplary differentiation agents include, but are not limited to:

-   -   1. Erythropoietin (Epo): It has been demonstrated that Epo        enhances NSC commitment to neuronal cell lineage and that this        can be used to treat mouse and rat models of stroke.    -   2. Brain derived neurotrophic factor (BDNF): BDNF is a known        survival factor and differentiation agent that promotes the        neuronal lineage.    -   3. Transforming growth factor beta and bone morphogenetic        proteins (BMPs): BMPs are known differentiation agents that        promote the neuronal lineage and the generation of specific        neuronal phenotypes (e.g.: sensory interneurons in the spinal        cord).    -   4. Thyroid hormone (TH, including both the T3 and T4 forms): TH        is known as a differentiation agent that promotes the maturation        and generation of oligodendroctyes. See, e.g., (Rodriguez-Pena,        1999).    -   5. Thyroid stimulating hormone (TSH) and Thyroid releasing        hormone (TRH): TSH/TRH promote the release of TH from the        anterior pituitary resulting in increased levels of circulating        TH. This agent could be used in combination with        pheromone/LH/hCG to promote oligodendrogliogenesis from NSCs.    -   6. Sonic hedgehog (SHH): SHH is a morphogen that patterns the        developing CNS during development and, in different        concentrations, promotes the generation of specific types of        neurons (eg: motoneurons in the spinal cord) and        oligodendrocytes. This agent could be used in combination with        pheromone/LH/hCG to promote neurogenesis and/or        oligodendrogliogenesis from NSCs.    -   7. Platelet derived growth factor (PDGF): PDGF promotes the        generation and differentiation of oligodendrocytes from NSCs.        This agent could be used in combination with pheromone/LH/hCG to        promote oligodendrogliogenesis from NSCs.    -   8. Cyclic AMP and agents which enhance the cAMP pathway, such as        pituitary adenylate cyclase activating polypeptide (PACAP) and        serotonin, are also good candidates for selectively promoting        neuron production.

Agents that can increase neural stem cell number include, without beinglimited to:

-   -   9. Follicle-stimulating hormone (FSH) often acts in concert with        LH; known to induce LH receptor expression and can therefore        enhance the effects of LH signaling.    -   10. Growth hormone (GH) can stimulate NSC proliferation.    -   11. Insulin growth factors (IGFs) are somatomedians that are        released from many tissues in response to GH and mediate many of        the growth promoting effects of GH. IGF-1 stimulates NSC        proliferation.    -   12. Growth hormone releasing hormone (GHRH) are secreted from        the hypothalamus and induces GH release from the anterior        pituitary, resulting in increased levels of circulating GH.    -   13. Prolactin (PRL) is secreted from the anterior pituitary and        known to promote NSC proliferation. PRL and pheromone/LH/hCG may        be used in combination to maximize NSC proliferation.    -   14. Prolactin releasing peptide (PRP) triggers the release of        prolactin and can be used in combination with pheromone/LH/hCG        to maximize NSC proliferation.    -   15. Fibroblast growth factor is a known mitogenic agent for        NSCs.    -   16. Estrogen is known to promote the proliferation of NSCs in        the hippocampus.    -   17. Serotonin is known to promote the proliferation of NSCs in        the hippocampus.    -   18. Epidermal growth factor is a known mitogenic agent for NSCs.    -   19. Transforming growth factor alpha (TGFalpha) is a known        mitogenic agent for NSCs.    -   20. Gonadotropin releasing hormone (GnRH) triggers the release        of LH and could be used in combination with or in place of        pheromone/LH/hCG to increase circulating levels of LH and        enhance NSC proliferation.    -   21. Ciliary neurotrophic factor and leukemia inhibitory factor:        Both of these agents, and others, signal via the gp130 subunit.        This signaling pathway has been demonstrated to promote NSC        self-renewal, thereby expanding the NSC population of the brain.        These agents could be used in combination with pheromone/LH/hCG        to promote NSC proliferation and increase the size of the NSC        population within the CNS.

Further provided by the present invention are methods of increasingneuron formation from neural stem cells in vitro or in vivo. Inparticular, methods of enhancing new olfactory neuron production areprovided.

The increase in neural stem cells or neurons is preferably at leastabout 10%, more preferably at least about 20%, even more preferably atleast about 30%, yet more preferably at least about 40%, still morepreferably at least about 50%, and further more preferably at leastabout 60%. Most preferably, the increase is at least about 80%.

The present invention also provides a method for treating orameliorating a neurodegenerative disease or condition in an animal,particularly a mammal. This can be achieved, for example, byadministering an effective amount of an LH and/or hCG to the mammal, ortransplanting to the mammal neural stem cells, progenitor cells derivedfrom neural stem cells, neurons and/or glial cells produced according tothe present invention. Preferably, neural stem cells are transplanted.In addition to the transplantation, LH/hCG and/or additional agents canbe further provided to the transplantation recipient, particularlyconcurrently with or after the transplantation.

One particularly interesting neurodegenerative condition is aging. Wehave found that the number of neural stem cells in the subventricularzone is significantly reduced in aged mice. Accordingly, it will be ofparticular interest to ameliorate problems associated with aging byincreasing neural stem cell numbers with pheromone/LH/hCG.

For example, the neural stem cell in the subventricular zone is thesource of olfactory neurons, and olfactory dysfunction is a hallmark offorebrain neurodegenerative diseases, such as Alzheimer's, Parkinson'sand Huntington's diseases. Disruption of neuronal migration to theolfactory bulb leads to deficits in olfactory discrimination, anddoubling the new olfactory interneuons enhances new odor memory(Rochefort et al., 2002). Therefore, pheromone/LH/hCG can be used toenhance olfactory discrimination or olfactory memory, as well asphysiological functions that are associated with olfaction and olfactorydiscrimination, such as mating, offspring recognition and rearing.

Another particularly important application of the present invention isthe treatment and/or amelioration of CNS injuries, such as stroke.

Compositions

The present invention provides compositions that comprise a pheromone,LH or hCG and optionally at least one additional agent. The additionalagent is capable of increasing neural stem cell number or enhancingneural stem cell differentiation to neurons or glial cells, as describedabove. The additional agent is preferably selected from the groupconsisting of follicle-stimulating hormone (FSH), gonadotropin releasinghormone (GnRH), prolactin, prolactin releasing peptide (PRP)erythropoietin, cyclic AMP, pituitary adenylate cyclase activatingpolypeptide (PACAP), serotonin, bone morphogenic protein (BMP),epidermal growth factor (EGF), transforming growth factor alpha(TGFalpha), transforming growth factor beta (TGFbeta), fibroblast growthfactor (FGF), estrogen, growth hormone, growth hormone releasinghormone, insulin-like growth factors, leukemia inhibitory factor,ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor(BDNF), thyroid hormone, thyroid stimulating hormone, sonic hedgehog(SHH), and/or platelet derived growth factor (PDGF). Most preferably,erythropoietin, prolactin, EGF and/or PACAP are added.

The pheromone can be any pheromone that is capable of increasing neuralstem cell numbers in the mammal. Assays for determining if a substanceis capable of increasing neural stem cell numbers are established in theart and described herein (e.g., see Examples 1 and 3). The pheromone ispreferably selected from the group consisting of2-sec-butyl-4,5-dihydrothiazole (SBT), 2,3-dehydro-exo-brevicomin (DHB),alpha and beta farnesenes, 6-hydroxy-6-methyl-3-heptanone, 2-heptanone,trans-5-hepten-2-one, trans-4-hepten-2-one, n-pentyl acetate,cis-2-penten-1-yl-acetate, 2,5-dimethylpyrazine, dodecyl propionate, and(Z)-7-dodecen-1-yl acetate (see, e.g., Dulac et al., 2003).

The LH/hCG useful in the present invention includes any LH or hCG analogor variant which is capable of increasing neural stem cell number. ALH/hCG analog or variant comprises a protein which contains at leastabout 30% of the amino acid sequence of at least one subunit of thenative human LH or hCG, and which possesses a biological activity of thenative LH or hCG. Preferably, the biological activity of LH or hCG isthe ability to bind the LH/hCG receptors. Specifically included asLH/hCG are the naturally occurring LH/hCG variants; LH/hCG counterpartsfrom various mammalian species, including but not limited to, human,other primates, rat, mouse, sheep, pig, and cattle; and the commonlyused analogs listed in Table 1 below. GnRH, or an analog thereof, can beused in the place of or in addition to LH/hCG.

TABLE 1 Common Analogs of GnRH, LH and hCG GnRH/LHRH agonists GnRHagonist, leuprorelin (pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt)Buserelin another LH-RH agonist Serophene: A prescription medicationthat initiates the release of GnRH LH Luveris ® (lutropin alfa) pureluteinizing hormone (recombinant human LH) hCG Ovidrel ®/Ovitrelle ®1(choriogonadotropin alfa); recombinant chorionic gonadotropin (r-hCG)Pregnyl ® is an injectable, highly purified preparation of humanchorionic gonadotropin obtained from the urine of pregnant women.Pregnyl has been in use throughout the world since 1932. NOVAREL ™(chorionic gonadotropin for injection, USP) Profasi: human chorionicgonadotropin (hCG). Profasi is administered intramuscularly.

Similarly, any additional compounds or agents that are useful in thepresent invention include their analogs and variants that share asubstantial similarity and at least one biological activity with thenative compounds or agents. For example, EGF can be used in conjunctionwith LH/hCG in the present invention. In addition to native EGF, an EGFanalog or variant can also be used, which should share a substantialamino acid sequence similarity with the native EGF, as well as at leastone biological activity with the native EGF, such as binding to the EGFreceptor. Particularly included as an EGF is the native EGF of anyspecies, TGFalpha, or recombinant modified EGF. Specific examplesinclude, but are not limited to, the recombinant modified EGF having adeletion of the two C-terminal amino acids and a neutral amino acidsubstitution at position 51 (particularly EGF51gln51; U.S. PatentApplication Publication No. 20020098178A1), the EGF mutein (EGF-X₁₆) inwhich the His residue at position 16 is replaced with a neutral oracidic amino acid (U.S. Pat. No. 6,191,106), the 52-amino acid deletionmutant of EGF which lacks the amino terminal residue of the native EGF(EGF-D), the EGF deletion mutant in which the N-terminal residue as wellas the two C-terminal residues (Arg-Leu) are deleted (EGF-B), the EGF-Din which the Met residue at position 21 is oxidized (EGF-C), the EGF-Bin which the Met residue at position 21 is oxidized (EGF-A),heparin-binding EGF-like growth factor (HB-EGF), betacellulin,amphiregulin, neuregulin, or a fusion protein comprising any of theabove. Other useful EGF analogs or variants are described in U.S. PatentApplication Publication No. 20020098178A1, and U.S. Pat. Nos. 6,191,106and 5,547,935.

As another example, PACAP can also be used in conjunction with LH/hCG.Useful PACAP analogs and variants include, without being limited to, the38 amino acid and the 27 amino acid variants of PACAP (PACAP38 andPACAP27, respectively), and the analogs and variants disclosed in, e.g.,U.S. Pat. Nos. 5,128,242; 5,198,542; 5,208,320; 5,326,860; 5,623,050;5,801,147 and 6,242,563.

Erythropoietin analogs and variants are disclosed, for example, in U.S.Pat. Nos. 6,048,971 and 5,614,184.

Further contemplated in the present invention are functional agonists ofLH/hCG or additional agents useful in the present invention. Thesefunctional agonists bind to and activate the receptor of the nativeagent, although they do not necessarily share a substantial sequencesimilarity with the native agent. For example, maxadilan is apolypeptide that acts as a specific agonist of the PACAP type-1 receptor(Moro et al., 1997).

Functional agonists of EPO have been extensively studied. EMP1 (EPOmimetic peptide 1) is one of the EPO mimetics described in Johnson etal., 2000. Short peptide mimetics of EPO are described in, e.g.,Wrighton et al., 1996 and U.S. Pat. No. 5,773,569. Small molecular EPOmimetics are disclosed in, e.g., Kaushansky, 2001. Antibodies thatactivate the EPO receptor are described in, e.g., U.S. Pat. No.5,885,574; WO 96/40231 and WO 97/48729).

Antibodies that have agonist activities for the EGF receptor aredescribed, e.g., in Fernandez-Pol, 1985 and U.S. Pat. No. 5,723,115. Inaddition, activating amino acid sequences are also disclosed in U.S.Pat. No. 6,333,031 for the EPO receptor, EGF receptor, prolactinreceptor and many other cell surface receptors; metal complexed receptorligands with agonist activities for the prolactin and EPO receptors canbe found in U.S. Pat. No. 6,413,952. Other methods of identifying andpreparing ligands for receptors, e.g., EPO and prolactin receptors, aredescribed, for example, in U.S. Pat. Nos. 5,506,107 and 5,837,460.

Commonly used analogs of certain additional agents can also be found inTable 2 below:

TABLE 2 Common Analogs of Additional Agents FSH Follitropin beta;Follistim/Puregon ®, recombinant follicle-stimulating hormone (FSH),pure gonadotropin widely used to treat infertility; launched by Organonin 1996 GONAL-f ™ (follitropin alpha) is recombinant humanfollicle-stimulating hormone, which is equivalent in its structure tothe naturally occurring human FSH in the body. BRAVELLE ™(urofollitropin for injection, purified); highly purified human-derivedFSH (Hfsh) only human-derived FSH approved for both subcutaneous (SC)and intramuscular (IM) injection. PRP (prolactin releasing peptide) hPRPSer-Arg-Thr-His-Arg-His-Ser-Met-Glu-Ile-Arg-Thr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Ser-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2 LIF Emfilermin(r-LIF) embryo implantation failure: still in clinical studies (NOT YETAPPROVED) EPO NeoRecormon; Erythropoietin beta; Roche epoetin omega;Baxter International Inc.; physicochemical characteristics differentfrom other erythropoietins or Epos (alpha and beta); currently approvedfor sale in 15 countries outside of the United States and WesternEurope. darbepoietin TH Armour Thyroid, natural desiccated thyroidhormone replacement drug, Forest Pharmaceuticals Cytomel, syntheticliothyronine sodium (T3), King Pharmaceuticals Levothroid, syntheticlevothyroxine, Forest Pharmaceuticals (currently not FDA approved as ofDecember 2003) Levoxyl, synthetic levothyroxine, from KingPharmaceuticals Nature-throid and Westhroid, natural desiccated thyroidhormone replacement drug, Western Research Laboratories Synthroid,synthetic levothyroxine, from Abbott Laboratories Thyrolar, syntheticliotrix, a combination of L-triiodothyronine (T3) and levothyroxinesodium (T4) Unithroid, synthetic levothyroxine, from Jerome StevensPharmaceuticals TSH Thyrogen, a synthetic thyroid stimulating hormone(TSH) for use in thyroid cancer patients, from Genzyme Pharmaceuticals,currently FDA approved TRH (thyroid releasing hormone) pGlu-His-ProAmide THYREL ® TRH (protirelin)

It should be noted that the effective amount of each analog, variant orfunctional agonist may be different from that for the native agent orcompound, and the effective amount in each case can be determined by aperson of ordinary skill in the art according to the disclosure herein.Preferably, the native agents, or analogs and variants that sharesubstantial sequence similarity with the native agents, are used in thepresent invention.

Pharmaceutical compositions are also provided, comprising an LH/hCG, anadditional agent as described above, and a pharmaceutically acceptableexcipient and/or carrier.

The pharmaceutical compositions can be delivered via any route known inthe art, such as parenterally, intrathecally, intravascularly,intravenously, intramuscularly, transdermally, intradermally,subcutaneously, intranasally, topically, orally, rectally, vaginally,pulmonarily or intraperitoneally. For example, it is shown in Example 6that intramuscular injection is an efficient route of delivering hCG toexert its function in the brain. Preferably, the composition isdelivered into the central nervous system by injection or infusion. Morepreferably it is delivered into a ventricle of the brain, particularlythe lateral ventricle. Alternatively, the composition is preferablydelivered by systemic routes, such as subcutaneous administration. Forexample, it has been discovered that prolactin, growth hormone, IGF-1,PACAP and EPO can be effectively delivered by subcutaneousadministration to modulate the number of neural stem cells in thesubventricular zone.

When the composition is not directly delivered into the brain, andmolecules in the composition do not readily cross the blood brainbarrier, a blood brain barrier permeabilizer can be optionally includedto facilitate entry into the brain. Blood brain barrier permeabilizersare known in the art and include, by way of example, bradykinin and thebradykinin agonists described in U.S. Pat. Nos. 5,686,416; 5,506,206 and5,268,164 (such asNH₂-arginine-proline-hydroxyproxyproline-glycine-thienylalanine-serine-proline-4-Me-tyrosine.psi.(CH₂NH)-arginine-COOH).Alternatively, the molecules can be conjugated to the transferrinreceptor antibodies as described in U.S. Pat. No. 6,329,508; 6,015,555;5,833,988 or 5,527,527. The molecules can also be delivered as a fusionprotein comprising the molecule and a ligand that is reactive with abrain capillary endothelial cell receptor, such as the transferrinreceptor (see, e.g., U.S. Pat. No. 5,977,307).

The pharmaceutical compositions can be prepared by mixing the desiredtherapeutic agents with an appropriate vehicle suitable for the intendedroute of administration. In making the pharmaceutical compositions ofthis invention, the therapeutic agents are usually mixed with anexcipient, diluted by an excipient or enclosed within such a carrierwhich can be in the form of a capsule, sachet, paper or other container.When the pharmaceutically acceptable excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the therapeutic agent. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the therapeutic agents, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

Some examples of suitable excipients include artificial cerebral spinalfluid, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,sterile water, syrup, and methyl cellulose. The formulations canadditionally include: lubricating agents such as talc, magnesiumstearate, and mineral oil; wetting agents; emulsifying and suspendingagents; preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the therapeutic agents after administration to the patient byemploying procedures known in the art.

For preparing solid compositions such as tablets, the therapeutic agentis mixed with a pharmaceutical excipient to form a solid preformulationcomposition containing a homogeneous mixture of a compound of thepresent invention. When referring to these preformulation compositionsas homogeneous, it is meant that the therapeutic agents are dispersedevenly throughout the composition so that the composition may be readilysubdivided into equally effective unit dosage forms such as tablets,pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. The compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

Another formulation employed in the methods of the present inventionemploys transdermal delivery devices (“patches”). Such transdermalpatches may be used to provide continuous or discontinuous infusion ofthe therapeutic agent of the present invention in controlled amounts.The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, for example, U.S.Pat. No. 5,023,252, herein incorporated by reference. Such patches maybe constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences.

The following examples are offered to illustrate this invention and arenot to be construed in any way as limiting the scope of the presentinvention.

EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. Abbreviations not defined have their generally acceptedmeanings.

TUNEL=Terminal deoxynucleotidyltransferase-mediated dUTP nick endlabeling° C.=degree Celsiushr=hourmin=minuteμM=micromolarmM=millimolarM=molar ml milliliterμl=microlitermg=milligramμg=microgramFBS=fetal bovine serumPBS=phosphate buffered salineDMEM=Dulbecco's modified Eagle's mediumMEM=modified Eagle's mediumEGF=epidermal growth factorNSC=neural stem cellSVZ=subventricular zonePACAP=pituitary adenylate cyclase activating polypeptidecAMP=cyclic AMPBMP=bone morphogenetic proteinCSF=cerebral spinal fluid

Materials and Methods

Female Mice Exposed to Male Mice Odor

Mice (CD1, 10 weeks old) were continuously exposed to the odor of theopposite gender or the same gender over a two-week time course. A socialinteraction component was not part of this study. Instead the mice wereonly exposed to the odors of the opposite gender. This was necessary tocontrol for confounds that would arise if done otherwise. For example,if males were placed together with females this would give the animalsthe opportunity to mate. Fowler's study on prairie voles (2002) hasshown increased neurogenesis in pregnant female voles, and even moresignificantly, the study of Shingo et al. (2003) showed that bothpregnancy and mating alone could result in an increase in neurogenesis.Therefore, if a neurogenic effect was seen it would be impossible toconclude that it was mediated by odor exposure alone.

With this in mind, a two-week continuous exposure protocol wasestablished to conduct this study, where the duration of the time coursewas chosen to account for the variable nature of neuronal cellproliferation in different environmental conditions. This also ensuredthat a surge in neuronal progenitor cell proliferation would not beoverlooked because previous behavioral studies have shown increases inneuronal progenitor cells varying from a one-day period to a two-weekperiod (Kempermann and Gage, 1999; Fowler et al., 2002).

Briefly, male mice were placed in a clean cage for two days. The malemice were then removed from the male odorized cage, and female mice werehoused in the cage for a desired length of time.

A total of 18 female (CD-1, 10 week old) mice were first chosen toexperience a continuous exposure to male (CD-1, 10 week old) or femalemice odor. Of the 18, 3 were randomly assigned to be exposed for 2 daysto male odor. Another 3 were chosen to participate in the 7-day maleodor exposure and another 3 in the 14-day male odor exposure condition.Similarly, an additional 3 were randomly assigned to experience 2 daysof female odor. Another 3 were chosen to participate in the 7-day femaleodor exposure condition and the remaining 3 were placed in the 14-dayfemale odor exposure condition.

Thus, in the first step, 9 male mice were placed into clean cages for 2days. After the males odorized their cages for two days, they weretransferred to a new clean cage. Then the females were transferred tothe male odorized cage for 2 days to experience the odor of the oppositegender. For the females assigned to the 2-day exposure the time coursewas complete; however those assigned to the 7 and 14 day exposures wouldhave to repeat the sequence, essentially being transferred to a freshodorized cage odorized by the same males to complete their time course.

To compare the effects of whether exposing females to opposite genderodor over the time course may have on neurogenesis, the remaining 9female mice selected above were to also experience a continuous genderodor, but odor of the same gender, under the same schematic as outlinedabove.

Upon the termination date of a group's time course, BrdU injections weregiven to the mice to label proliferating cells in the SVZ. Furtherimmunohistochemical analysis were done and are outlined below.

Immunohistochemistry

To examine the number of progenitor cells in the SVZ after treatment,bromodeoxyuridine (BrdU) injections were used to label these cells.Animals received BrdU injections (120 mg/kg, i.p., dissolved in 0.007%NaOH in phosphate buffer) every 2 hours for a 10 hour period. Eachinjection of BrdU will only label proliferating cells in the S-phase andthe purpose of having a series of BrdU injection is to ensure thecontinuing availability of BrdU for full incorporation (Morshead and vander Kooy, 1992).

The animals were sacrificed by anaesthetic overdose and perfusedtranscardially with 4% paraformaldehyde in PBS, pH 7.2. Brains werepost-fixed in the same paraformaldehyde solution overnight at 4° C., andcyroprotected for 24 hours in 20% sucrose in PBS. The brains were thenembedded in Tissue Tek O.C.T. compound (Sakura Fineteck, Torrance,Calif.) before being cyrosectioned at 14 μm.

The antibodies used for staining were rat anti-BrdU and guinea piganti-DCX.

The sections were post-fixed with acetone for 30 seconds at roomtemperature and washed with PBS. For BrdU staining, the tissue wastreated with 1M HCl for 22 minutes at 60° C. to denature cellular DNA.Sections were then incubated for 24 hours at room temperature in primaryantibody (rat anti-BrdU, 1:50) diluted in 0.3% PBS-T containing 10% NGS,washed with PBS, and then incubated with goat-anti-rat secondaryantibodies conjugated to biotin (1:200) for 1 hour at room temperature.After rinsing with ddH₂O, sections were mounted with Fluorosave, becausestaining was visualized with CY3-Streptavodin, and viewed with a ZeissAxiophot fluorescence microscope.

For DCX staining, sections were incubated for 24 hrs at room temperaturein primary antibody (goat anti-DCX, 1:500) diluted in 0.3% PBS-Tcontaining 10% NGS, washed with PBS, and then incubated with donkeyanti-goat biotin secondary antibody for 1 hour at room temperature.After rinsing, an amplification procedure was performed by washing theslides with PBS and incubating them with CY3-Streptavodin and Hoechstfor 1 hour at room temperature. After rinsing with ddH₂0, sections weremounted with Fluorosave and viewed with a Zeiss Axiophot.

Quantification of Immunohistochemistry Results

BrdU in the SVZ: A one-in-ten series of coronal sections (14 μm) fromthe rostral tip of the lateral ventricle to caudal most aspect of theventricles (total 10 sections) were collected. BrdU-positive cells werethen counted in the defining ependymal-subependymal layer.

DCX in the dorsolateral corner of the SVZ: A one-in ten series ofcoronal sections (14 μm) from the rostral tip of the lateral ventricleto 980 μm caudal of the ventricles (total 10 sections) was performed.DCX-positive cells were then counted in the dorsolateral corner.

Male Mice Exposed to Female Mice Odor

To see if opposite gender odor had any effect of male mice, the samemethodology was used to continuously expose male mice to female miceodor. The identical time course of 2-day, 7-day, and 14-day odorexposure to female mice was used, as well as a 2-day, 7-day, and 14-dayodor exposure to male mice for comparison. Immunohistochemical andquantification components were also identical to the design for thefemales.

Neural Stem Cell Culture and Growth Factors

Generation and differentiation of spheres from embryonic and adultforebrain were performed as described previously with minormodifications (Reynolds and Weiss, 1992; Reynolds et al., 1992).Briefly, striato-pallidum complexes were removed from mouse embryos atE14 and collected into PBS containing 0.6% glucose, penicillin (50U/ml), and streptomycin (50 U/ml; both from Life Technologies,Gaithersburg, Md.) and then transferred into the standard culture mediumcomposed of DMEM-F-12 (1:1), glucose (0.6%), glutamine (2 mM), sodiumbicarbonate (3 mM), HEPES buffer (5 mM), insulin (25 μg/ml), transferrin(100 μg/ml), progesterone (20 nM), putrescine (60 μM), and seleniumchloride (30 nM) (all from Sigma, St. Louis, Mo., except glutamine fromLife Technologies). For adult neural stem cell cultures, medial andlateral portions of the lateral ventricle subependyma from the adultbrain were dissected from both hemispheres, pooled together,subsequently cut into 1 mm² fragments, and transferred into the standardculture medium containing 1.33 mg/ml trypsin, 0.67 mg/ml hyaluronidase,and 0.2 mg/ml kynurenic acid (all from Sigma). After 30 min at 37° C.,the tissue was transferred to the standard culture medium containing 0.7mg/ml trypsin inhibitor (Roche Diagnostics, Laval, Quebec, Canada).Tissue pieces were mechanically dissociated with micropipettes. Cellswere seeded at various densities into the standard culture medium, whichalso contained EGF (20 ng/ml human recombinant; Peprotech, Rocky Hill,N.J.). Cells were cultured for 7 days in vitro (DIV) and formed floatingcell clusters (spheres). All the mice for culture experiments werekilled by cervical dislocation.

Implantation of the Osmotic Pumps and Growth Factor Infusion

Sixteen 8-week-old CD-1 mice (Charles-River, Laval, Quebec, Canada) wereanesthetized with sodium pentobarbital (120 mg/kg, i.p.) and implantedwith osmotic pumps (Alzet 1007D; Alza, Palo Alto, Calif.). The cannulaswere located in the right lateral ventricle (anteroposterior +0.2 mm,lateral +0.8 mm to bregma, and dorsoventral 2.5 mm below dura with theskull leveled between lambda and bregma). LH (33 μg/ml human LH derivedfrom the pituitary; the National hormone and peptide program, Universityof California Los Angeles, Calif., USA) was dissolved in 0.9% salinecontaining 1 mg/ml mouse serum albumin (Sigma). Each animal was infusedfor 6 d with either vehicle alone or LH at a flow rate of 0.5 μl/hr,resulting in a delivery of 400 ng/d of LH.

The TUNEL Assay

TUNEL labeling was performed using the ROCHE In Situ Cell Death Kit (cat#1 684 795) according to the manufacturers instructions for use onfrozen tissue.

Example 1 Odors of the Opposite Gender Stimulates Proliferation

To determine the impact of male or female odors, adult mice were exposedto the odors of the opposite gender for 2 days, 7 days or 14 days. Acontrol group was exposed to the odors of the same gender for the sameperiod of time. The mice then received BrdU to label proliferatingcells, and the locations of the BrdU positive cells were identified byimmunohistochemical studies. Female or male mice were also exposed tocontrol unodorized cages in parallel experiments for 7 days (female) or2 days (male), and these animals did not differ from animals exposed tosame sex odors (data not shown)

As shown in FIG. 1A, proliferating cells in the SVZ of female miceremained at the same level after being exposed to female odors for 2, 7or 14 days. In the female group exposed to male odors, however,proliferating cells in the SVZ changed with time: increasedsignificantly after 7 days and decreased significantly after 14 days. A2-day exposure had no significant effect. The same pattern was observedwhen Ki67 was used to label proliferating cells (FIG. 1B), indicatingthat the change in BrdU positive cells reflected a change ofproliferation level rather than preferred uptake of BrdU.

The male and female mice used in the above experiments were from thesame litter or previously shipped together. Therefore, they had beenexposed to the odor of one another before the experiments wereperformed. To rule out the possibility that this effect was specific tolittermates or animals that have been pre-exposed to the odor ofinterest, mice from different litters that had not previously been inthe same place were used to repeat the experiments. Similar effects wereobserved whether the mice were littermates or not (FIG. 1C), indicatingthat the effects of the male odor are not limited to particular littersor pre-exposure of the odor.

Female odors also affected proliferation in male mouse brains, but in adifferent temporal pattern. When males were exposed to female odors for2 days, there was a sudden increase in the number of BrdU positive cells(FIG. 2A) or Ki67 positive cells (FIG. 2B). After a 7 or 14 dayexposure, however, the number of newly proliferated cells decreased tothe control level. As with the female mice, the effects of female odorscould be observed when different litters were used (FIG. 2C).

Strikingly, the neural stem cells in the hippocampus also responded togender-specific odors. Again, exposure for two days to male odors had nosignificant effects on female mice, but a 7-day exposure resulted in asignificant increase in proliferation in the hippocampus (FIG. 3). Afteran exposure for 14 days, levels of proliferating cells weresignificantly lower in females exposed to male odors when compared withthe females that had been exposed to female odors. To our knowledge,this is the first time that any stimulus, other than growth factors(e.g., EGF plus FGF), is shown to exert the same effects on the neuralstem cells in the SVZ and the hippocampus.

As an additional control, female mice were exposed to odors of castratedmale mice for 7 days. The results show that the numbers of BrdU labeledcells in either the SVZ or hippocampus did not increase in thesefemales, as compared to females that had been exposed to sham maleodors. Sham male odors are odorized cages that were odorized by malemice that underwent a sham castration surgery. Females exposed to theodors of these sham castrated males showed sex pheromone-inducedneurogenesis, however females exposed to the odors of castrated malesdid not show increased neurogenesis. Similarly, male mice exposed to theodors of adrenalectomized females for two days also showed no increasein the number of BrdU labeled cells in the SVZ or the hippocampus, ascompared to males exposed to sham female odors. Castration andadrenalectomy are known to reduce pheromone levels (Ma et al., 1998;Kiyokawa et al., 2004; Zhang J. et al. (2001)). These results thusfurther support the observation that pheromones induce neural stem cellproliferation in both the SVZ and hippocampus of the opposite gender.

Example 2 Odors of the Opposite Gender Stimulates Neurogenesis but notCell Survival

Neurogenesis was also enhanced upon exposure to the odors of theopposite gender. Thus, tissue sections from Example 1 were stained fordoublecortin, a cytoplasmic protein expressed in neuronal progenitorcells, to determine the extent of neurogenesis in the mice describedabove. As in the case of proliferating cells, female mice hadsignificantly more doublecortin positive cells after a 7-day exposure tomale odors (FIG. 4) while male mice had significantly more doublecortinpositive cells after a 2-day exposure to female odors (FIG. 5).

To determine if pheromones from the opposite gender also impact survivalof neural cells, the TUNEL assay was performed. The results indicatethat no significant difference can be observed in the SVZ (FIG. 6A) orolfactory bulb (FIG. 6B) of female mice after a 7-day exposure to maleodors.

Example 3 The Effects of LH In Vivo and In Vitro

Male pheromones are known to increase the levels of the luteinizinghormone

(LH) and decrease the levels of prolactin, while female pheromones areassociated with an increase of prolactin (Dulac et al., 2003). In anattempt to investigate how pheromones enhance neural stem cellproliferation and neurogenesis in the opposite gender, animals wereinfused with LH as described in Materials and Methods. Indeed, LHincrease proliferation significantly in the SVZ of both female (FIGS. 7Aand 7B) and male mice (FIG. 8).

To confirm that LH stimulates proliferation of neural stem cells, neuralstem cell cultures were established as described in Materials andMethods. Primary spheres were dissociated and plated in the presence ofEGF or EGF plus LH (30 nM) at limited density to allow formation ofsecondary spheres. The number of secondary spheres were counted, and theresults are shown below:

Neural Stem Cells Isolated from Female Mice:

EGF EGF + LH Exp. #1 172.3 255.9 Exp. #2 157.6 241.9 Exp. #3 197.9 258.9

Neural Stem Cells Isolated from Male Mice:

EGF EGF + LH Exp. #1 144.4 168.6 Exp. #2 225.1 275.1 Exp. #3 168.2 195.9

Thus, LH is also capable of increasing self-renewal of neural stem cellsin culture, and it is more effective on the neural stem cells isolatedfrom female mice than those isolated from male mice.

Example 4 The Effects of hCG In Vivo and In Vitro

HCG has the same activity as LH. When mice are infused with arecombinant hCG (choriogonadotropin alfa; Ovidrel®/Ovitrelle®1) orvehicle alone according to the protocol described in Materials andMethods, it is discovered that hCG significantly increases proliferationin the SVZ in both male and female mice. Proliferating cells in thehippocampus also increase significantly in both gender.

Neurogenesis is also assessed by using a neuron marker, doublecortin orNeuN. The number of doublecortin or NeuN positive cells in the SVZ orolfactory bulb is significantly higher in the mice infused with hCG.

To confirm that hCG stimulates proliferation of neural stem cells,neural stem cell cultures are established as described in Materials andMethods. Primary spheres are dissociated and plated in the presence ofEGF or EGF plus choriogonadotropin alfa at limited density to allowformation of secondary spheres. The number of secondary spheres are thencounted, and the results indicate that hCG significantly increases thenumber of secondary spheres whether the neural stem cells are from maleor female animals.

Example 5 The Effects of Additional Agents

An additional agent, prolactin, is included in the experiments describedin Example 3 or 4. Thus, mice are infused with

(1) a combination of LH and prolactin or hCG and prolactin;

(2) LH or hCG; or

(3) vehicle alone (control).

The results show that while LH or hCG increases proliferation in the SVZas compared to the control group, the addition of prolactin furtherenhances the effects of LH or hCG. Similarly, when prolactin is addedwith LH or hCG in neural stem cell cultures, self-renewal (the number ofsecondary spheres from primary spheres) is enhanced.

Similarly, Epo (NeoRecormon) is included with LH or hCG to determine itseffects on neurogenesis, and the results show that Epo enhanced thenumber of doublecortin positive cells over the level achieved by LH orhCG alone.

Although the Examples described above employ specific agents, it shouldbe noted that any analog or variant of LH/hCG, including the compoundslisted in Table 1, can be used as LH or hCG. Similarly, any additionalagent capable of enhancing neurogenesis and analog/variant thereof,including those listed in Table 2, can be used in Example 5 in the placeof NeoRecormon. Glial cell formation can be practiced using the methodsdescribed herein and knowledge available in the art.

Example 6 Intramuscular Delivery of hCG

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) arecommercially available drugs for human use, marketed as Pregnyl andProfasi, respectively. The maximal safe dose for each of these drugs is10,000 USP units per day via intramuscular injection. This correspondsto approximately a 5.0 USP unit dose for a mouse of 30 grams. To testwhether this dose would be sufficient to induce forebrain neurogenesisin mice, we performed the following experiment.

Six to eight week old female CD-1 mice received a single intramuscularinjection of 5.0 USP units of recombinant hCG (Sigma Catalog Number C6322) in a 0.05 ml volume (diluted in saline). Control mice received asingle injection of saline alone. The mice then received 6 injections ofBrdU (120 mg/kg), one every two hours, beginning two hours after the hCGinjection. The mice were sacrificed thirty-minutes after the last BrdUinjection, perfused transcardially with 4% paraformaldehyde, and thetissue was processed for cryosectioning. Brains were sectioned seriallyat 14 microns onto two sets of seven slides each, 12 sections on eachslide. The number of BrdU positive cells was counted in the forebrainSVZ on one slide for each of the control and hCG injected animals. Thefollowing data are the average numbers of BrdU+ cells per section in thecontrol (saline only) and hCG-injected mice, respectively.

Saline: 163±6 (n=4)

hCG: 206±13 (n=4; *p<0.02; paired t-test)

Therefore, a single low dose hCG injection increased proliferation inthe forebrain SVZ by 26%.

Example 7 LH Receptor Knock-Out Mice Experiments

To investigate whether the LH receptor directly mediates the effects ofLH on the neural stem cells in the SVZ and hippocampus, we determinedthe levels of LH receptors in both areas using immunohistochemicalanalyses. The results indicate that LH receptors can be found in bothSVZ and hippocampus in male and female mice, although the males hadlower levels of LH receptors compared to the females. Thus, LH probablybinds directly to its receptors in the SVZ and hippocampus to triggerthe biological functions described herein.

To further investigate the role of LH, LH receptor (LHR) knock-out (KO)mice were used in the odor-exposure experiments as described inExample 1. The mice were previously described in Zhang, F. P. et al.,2001, and Huhtaniemi et al., 2002. Eight to ten week old adult LHRwildtype (WT) and KO mice were exposed to the odors of the oppositegender for 2 or 7 days. On the 2nd (male exposed to female odor) and 7thday (female exposed to male odor) of exposure the animals received 6injections of BrdU (120 mg/kg), once every two hours. The mice were thensacrificed and transcardially perfused with 4% paraformaldehyde, about30 minutes following the last injection, and the tissue was processedfor cryosectioning. The forebrains of the mice were seriallycryosectioned at 14 microns onto 7 slides, with 10 sections on eachslide. A single slide from each animal was then immunostained for BrdU,and the total number of BrdU positive cells in the SVZ was quantified.Mice that were exposed to unodorized cages are used as baselinecontrols.

The results are shown in FIGS. 9 and 10. As expected, male odor resultedin an increase of proliferation in both the SVZ and hippocampus offemale wild type mice (+/+) (FIGS. 9A and 9B). In the LHR knock-out mice(−/−), however, no increase of proliferation was observed in thehippocampus after exposure to male odor (FIG. 9B). These resultsindicate that LH receptor signaling is important for the effects of malepheromones in the hippocampus of female mice. Interestingly, the lack ofLH receptor did not affect proliferation in the SVZ in response to malepheromones (FIG. 9A). Similarly, in male mice, LH receptor knock-out hadno impact on female pheromone-induced proliferation in either the SVZ orhippocampus (FIG. 10). Thus, although LH is sufficient to induce neuralstem cell proliferation in the SVZ and hippocampus in both females andmales, there is a factor (or factors) that can also mediate the actionsof pheromones in the SVZ and the male hippocampus.

1-9. (canceled)
 10. A method of enhancing neurogenesis in a mammalhaving or suspected of having a central nervous system (CNS) injury,comprising administering an effective amount of a luteinizing hormone(LH) or a human chorionic gonadotrophin (hCG) and an erythropoietin(EPO) to the mammal, wherein neurogenesis is enhanced in the mammal. 11.The method of claim 10 wherein the mammal is an adult.
 12. The method ofclaim 10 wherein the neurogenesis occurs in the subventricular zone. 13.The method of claim 10 wherein the neurogenesis occurs in thehippocampus.
 14. The method of claim 10 wherein the LH or hCG and EPOare administered systemically.
 15. The method of claim 10 wherein the LHor hCG and EPO are administered to the brain of the mammal. 16-25.(canceled)
 26. The method of claim 10 wherein the CNS injury is selectedfrom the group consisting of stroke, traumatic brain injury and spinalcord injury.
 27. The method of claim 26 wherein the CNS injury isstroke.
 28. The method of claim 26 wherein the CNS injury is traumaticbrain injury.
 29. The method of claim 26 wherein the CNS injury isspinal cord injury.
 30. A method of reducing a symptom of a CNS injuryin a mammal, comprising administering an effective amount of an hCG oran LH to the mammal.
 31. The method of claim 30 wherein the CNS injuryis selected from the group consisting of stroke, traumatic brain injuryand spinal cord injury.
 32. The method of claim 31 wherein the CNSinjury is stroke.
 33. The method of claim 31 wherein the CNS injury istraumatic brain injury.
 34. The method of claim 31 wherein the CNSinjury is spinal cord injury.
 35. The method of claim 30 furthercomprising administering an effective amount of an erythropoietin (EPO)to the mammal.
 36. The method of claim 35 wherein the hCG or LH isadministered to the mammal prior to the administration of the EPO to themammal.
 37. The method of claim 36 wherein the hCG or LH and EPO areadministered systemically.